The introduction of antibotics in the 1930's provided an effective method for the treatment and control of infectious diseases not before equaled in the history of medicine. However, along with its successes it also created a novel medical problem, namely serious side effects due to the disruption of the ecological balance among organisms of the normal flora of the body by the antibiotic. This imbalance often allows organisms with disease producing potential to grow more freely and to realize their pathogenic potential. Pneumonia caused by pencillin resistant staphylococci is a well known example of this condition. Such is the case with the anaerobic bacterium, Clostridium difficile, which lives normally in the gut of many humans as a non-troublesome parasite. Members of this clostridial species are relatively resistant to the antibiotic clindamycin and to related drugs. In contrast, many other bacteria of the normal gut are killed or their growth severely repressed by the same antibiotic. C. difficile produces a toxin which when present in sufficient amount causes pseudomembranous colitis in patients being treated. This species of clostridium and its toxin have not yet been characterized. The research described in this proposal has as its principal objectives to produce, isolate and purify the toxin of C. difficile and to develop a specific and sensitive assay for it. My research plan includes: 1) the determination of optimal growth conditions for production of the amounts of toxin necessary for this work 2) purification of the toxin using isoelectric focusing, gel filtration and other standard methods, 3) production of antitoxin specific for the toxin and 4) development of an assay using the antitoxin, to detect specifically small amounts of toxin in the presence of other proteins. This assay will be used to determine factors which influence the synthesis of toxin by C. difficile during its growth. It will also be suitable for use as a diagnostic test in clinical medicine. Accomplishment of these goals will lead directly to biochemical and biophysical characterization of the toxin to a determination of ecologic and genetic factors which determine production of the C. difficile toxin to a determination of ecologic and genetic factors which determine production of the C. difficile toxin and eventually to elucidation of its mode of action at a molecular level.